6.3. Jurkat-APC Coculture

Coculture with Antigen-Presenting Cells (APCs)

1. Prepare cell suspension of Jurkat reporter cells expressing peptide-specific TCR at 1 × 10⁶ cells/ml in complete RPMI1640 medium, and aliquot 100 μl into one well of a 96-well plate.
2. Prepare cell suspension of APCs (See Cellecta Cat.# YK562-MH-A0201-P for more information about how to make peptide loaded engineered APCs) at 1 × 10⁶ cells/ml in complete RPMI1640 medium.
3. Add 100 μl APCs (step 2) into each well with aliquoted Jurkat reporter cells (step 1), and mix well.
4. Incubate mixed cells at 37°C, 5%CO₂.
5. At 20 hours post-coculture, collect each cell mixture and put it into 500 μl of FACS buffer.
6. Perform FACS analysis for the GFP population, which reflects the activation of these Jurkat reporter cells.

Please refer to Jurkat Reporter Cells and K562 Antigen Presenting Cells for more information about these cell-lines.